KR102453319B1 - Fermented extract for antioxidant, anti-inflammatory, whitening, and anti-wrinkle using Lactobacillus kunkeei Hankook-001strain, and manufacturing method of it, and cosmetic composition containing fermented extract - Google Patents

Abstract

The present invention relates to a fermented extract for anti-oxidant, anti-inflammatory, whitening, anti-wrinkle effects using Lactobacillus kunkeei Hankook-001 strain, a manufacturing method thereof, and a cosmetic composition containing the fermented extract for anti-oxidant, anti-inflammatory, whitening, and anti-wrinkle effects and, more specifically, to a cosmetic composition containing a fermented extract obtained by extracting wild ginseng cultured roots and fermenting the same using the Lactobacillus kunkeei Hankook-001 strain (Accession Number: KCTC 14478BP), wherein the Lactobacillus kunkeei Hankook-001 strain provides anti-oxidant, anti-inflammatory, whitening, and anti-wrinkle effects, a manufacturing method thereof, and a cosmetic composition containing the fermented extract for anti-oxidant, anti-inflammatory, whitening, and anti-wrinkle effects.

Description

Fermented extract for antioxidant, anti-inflammatory, whitening, and anti-wrinkle using Lactobacillus kunki Hankook-001 strain and manufacturing method thereof, and antioxidant, anti-inflammatory, whitening, and anti-wrinkle cosmetic composition comprising the fermented extract {Fermented extract for antioxidant, anti- inflammatory, whitening, and anti-wrinkle using Lactobacillus kunkeei Hankook-001strain, and manufacturing method of it, and cosmetic composition containing fermented extract}

The present invention relates to a fermentation extract for antioxidant, anti-inflammatory, whitening, and anti-wrinkle using Lactobacillus kunki Hankook-001 strain and a method for preparing the same, and to a cosmetic composition for antioxidant, anti-inflammatory, whitening, and anti-wrinkle comprising the fermented extract, and more Specifically, by providing a cosmetic composition containing a fermented extract extracted from wild ginseng cultured roots and fermented using Lactobacillus kunkeei Hankook-001 strain (Accession No.: KCTC 14478BP), antioxidant, anti-inflammatory, whitening , Fermented extract for antioxidant, anti-inflammatory, whitening, anti-wrinkle using Lactobacillus kunki Hankook-001 strain that can provide anti-wrinkle effect and method for manufacturing the same, antioxidant, anti-inflammatory, whitening, anti-wrinkle containing the fermented extract It relates to a cosmetic composition.

Human skin is largely composed of the 'epithelium' that surrounds the outer surface of the skin, the 'dermis' that supplies the substrate supporting structures such as blood vessels and nerves under the epidermis, and the 'subcutaneous layer' containing a lot of fat between the dermis and the muscles. organization is made up of.

Among them, it is known that the dermis contains about 70% moisture and the epidermis contains about 30% moisture.

However, this is only the skin standard of a healthy person, and when the moisture content of the epidermis falls below 10%, the skin becomes rough and skin aging is accelerated.

In addition, as dry skin is maintained, various inflammations such as skin troubles and psoriasis and atopy may be induced.

On the other hand, lactic acid bacteria are bacteria that play a beneficial role in the human body and produce lactic acid (lactic acid) by fermenting sugars such as carbohydrates.

These lactic acid bacteria are known to have the physiological activity of enhancing immunity, especially gastrointestinal immunity, and according to the recent well-being trend, as a social atmosphere is created that good food is good to eat, various cosmetic materials beneficial to the body have been developed and lactic acid bacteria have been developed. of the culture medium has been reported to have effects such as moisturizing the skin, increasing the turnover rate of the stratum corneum, and relieving wrinkles.

For example, Korean Patent Application Laid-Open No. 1991-0019600 discloses a technology related to a lactic acid bacteria culture medium for cosmetics having moisturizing and active oxygen removal ability, and in the case of Korean Patent Application Laid-Open No. 2013-0096604, a fermented extract of germinated grains as a main component is disclosed. As a technology related to a fermented cosmetic and a method for manufacturing the same, a cosmetic composition containing a product produced by fermenting and culturing lactic acid bacteria, or using a fermented liquid obtained by fermenting germinated grains with lactic acid bacteria of a specific genus into a cosmetic is disclosed. .

And, in the case of Korean Patent Application Laid-Open No. 2013-0115943, a technology related to a composition for anti-inflammatory, skin whitening and anti-aging including a supernatant of lactic acid bacteria lysate is disclosed.

On the other hand, in general, as for the types of wild ginseng, it is said that the types of natural wild ginseng in the soil are classified into 18 types, such as thousand species and genus species, depending on the conditions of the soil's native habitat.

Cheonjong (natural wild ginseng), which is naturally born from the mountains and the seeds are dropped, wild animals or birds eat wild ginseng seeds and excreted in the mountains; There are many types of wild ginseng, such as Jangnoe ginseng (Gasam) cured with natural ginseng and Dongja ginseng, which is an old wild ginseng whose number of years is unknown.

In addition, it contains a large amount of physiologically active substances such as saponin, which other plants do not have at all. , brain function enhancement, aging suppression, and weak constitution improvement.

Wild ginseng cultured roots obtained from plant tissue culture were targeted because the extracts extracted from cultured roots of wild ginseng artificially had similar components and functions to those of natural wild ginseng.

As a method for making the medicinal ingredients of these wild ginseng cultured roots easily absorbed into the human body, and for strengthening the components present in trace amounts in ginseng, fermentation using lactic acid bacteria or enzyme treatment is used.

In this application, a cosmetic composition that can be usefully used for antioxidant, anti-inflammatory, whitening, and anti-wrinkle properties by using the Lactobacillus kunki Hankook-001 strain described above in line with the eco-friendly era was provided, and the effect as a cosmetic manufactured through this was verified. , thereby completing the present application by confirming that the cosmetic composition containing the Lactobacillus kunki Hankook-001 strain can provide antioxidant, anti-inflammatory, whitening, and anti-wrinkle effects.

Korean Patent Publication No. 1991-0019600 Korean Patent Publication No. 2013-0096604 Korean Patent Publication No. 2013-0115943

The present invention has been researched and developed to solve the above problems, and an object of the present invention is to provide a method for producing a fermented extract effective in antioxidant, anti-inflammatory, whitening, and anti-wrinkle.

Another object of the present invention is to provide a method for producing a fermented extract fermented with Lactobacillus kunkeei Hankook-001 (Accession No.: KCTC 14478BP), thereby providing an excellent cosmetic composition as a cosmetic raw material.

The present invention relates to a method for producing a fermented extract effective in antioxidant, anti-inflammatory, whitening, and anti-wrinkle, extracting cultured wild ginseng roots obtained by tissue culture to obtain a wild ginseng cultured root extract, and Lactobacillus kunki Hankook-001 (Lactobacillus kunkeei Hankook-001) strain is inoculated and fermented, and the sediment and lactic acid bacteria are removed to prepare a fermentation extract.

As can be seen through the present invention, a cosmetic composition containing a fermented extract fermented using a Lactobacillus kunkeei Hankook-001 (Accession No.: KCTC 14478BP) strain has antioxidant, anti-inflammatory, whitening, It exerts an anti-wrinkle effect.

1 is a 16S rRNA sequence diagram as a result of identification of Lactobacillus kunkeei Hankook-001 (Accession No.: KCTC 14478BP) of the present invention.
2 is a result confirming the free radical scavenging activity of the extracts or fermentation extracts of Examples 1 to 2.
3 is a result confirming the HDF cell viability of the extracts or fermentation extracts of Examples 1 to 2.
4 is a result confirming the B16F0 cell viability of the extracts or fermentation extracts of Examples 1 to 2.
5 is a result confirming the survival rate of RAW 264.7 cells of the extracts or fermentation extracts of Examples 1 to 2.
6 is a result of confirming the NO production inhibition of the extracts or fermentation extracts of Examples 1 to 2;
7 is a result of confirming the Melanin assay of the extracts or fermentation extracts of Examples 1 to 2.
8 is a result confirming the expression of MMP-1 in the extracts or fermentation extracts of Examples 1 to 2.
Figure 9 is a deposit certificate verifying the deposit of the present invention Lactobacillus kunkeei Hankook-001 ( Lactobacillus kunkeei Hankook-001) (Accession No.: KCTC 14478BP) strain.

Hereinafter, the present invention will be described.

The method for producing a fermented extract for antioxidant, anti-inflammatory, whitening, and anti-wrinkle according to the present invention,

The wild ginseng cultured root obtained by tissue culture was extracted with purified water to obtain an extract,

It is characterized in that the extract is inoculated with Lactobacillus kunki Hankook-001 of accession number KCTC 14478BP and fermented, and then the precipitate and lactic acid bacteria are removed to prepare a fermented extract.

The fermentation extract finally obtained in the present invention is preferably contained in an amount of 0.01 to 10.0% by weight based on the total cosmetic composition, and when it is contained in an amount of less than 0.01% by weight, the improvement effect on the skin is insignificant, and when it exceeds 10.0% by weight, Since it is not economical due to the low efficiency compared to the amount of material input, it is set to 0.01 ~ 10.0% through an experiment depending on the concentration.

In the present invention, the culture of the Lactobacillus kunkeei Hankook-001 (Accession No.: KCTC 14478BP) strain preferably adjusts the pH of the culture medium to 5.0 to 7.0, more specifically to 5.8, and the optimal culture time is 12 hours to 48 hours, more specifically 44 hours, the optimum culture temperature is characterized in that the culture at 27 to 37 ℃, more specifically 32 ℃.

In addition, the present invention relates to a basic cosmetic formulation in which the cosmetic composition is composed of a shampoo, a lotion, a gel, a water-soluble liquid, a cream, an essence, an oil-in-water (O/W) type and a water-in-oil (W/O) type;

a makeup base, foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, cheek color, and eyebrow pencil of a water-in-water or water-in-oil type makeup base; It is characterized in that any one selected from among.

As used herein, the term 'composition' preferably refers to a composition for external application for skin, and the composition for external application for skin generally refers to all compositions applied to the skin, and includes pharmaceuticals, quasi-drugs, cosmetics, and the like.

In addition, the blending amount of the active ingredient of the ferment extract of the present invention to the external preparation for skin is preferably 0.01 to 10.0%, more preferably 0.1 to 5.0%, based on the total amount of the composition.

On the other hand, in the present invention, "included as an active ingredient" means that the culture filtrate is added to the composition to the extent that it can exhibit the effect of improving the skin condition from the composition of the present invention, and for component delivery and stabilization, etc. It is meant to include formulating in various forms by adding various components as sub-components.

Hereinafter, the present invention will be described in more detail through examples. However, these Examples are only exemplary descriptions of the present invention, and the scope of the present invention is not limited to these Examples.

Example 1: Preparation of fermented wild ginseng cultured root extract

100 g of cultured wild ginseng root was placed in an extraction container, mixed with 1000 g of purified water, and extracted at 90° C. for 3 hours to prepare an extract, cooled and filtered.

Next, the mixed extract was sterilized at 121 °C for 15 minutes for incubation.

After the sterilization was finished, it was allowed to cool, inoculated with Lactobacillus kunki Hankook-001, and cultured and fermented at 35° C., 150 rpm, for 2 days to prepare a fermentation broth.

Then, the fermentation broth was centrifuged at 10,000 rpm for 20 minutes to obtain a fermented extract from which precipitates and lactic acid silica were removed.

This was concentrated under reduced pressure and vacuum dried to obtain a powder.

Example 2: Preparation of wild ginseng cultured root extract

100 g of cultured wild ginseng root was placed in an extraction container, mixed with 1000 g of purified water, and extracted at 90° C. for 3 hours to prepare an extract, cooled and filtered.

This was concentrated under reduced pressure and vacuum dried to obtain a powder.

Experimental Example 1: Free radical scavenging activity measurement experiment

In order to measure the free radical scavenging activity of each extract prepared in Examples 1 to 2, the free radical scavenging activity was measured using the DPPH method.

The DPPH method measures free radical scavenging activity by reducing power using a free radical called DPPH (2,2-Di(4-tert-octylphenyl)-1-1picrylhydrazyl free radical).

The degree of decrease in absorbance due to the reduction of DPPH by the test material was compared with the absorbance of the blank test solution, and the free radical scavenging rate was measured at a wavelength of 560 nm.

Extracts were prepared for each concentration in order to measure the DPPH free radical scavenging activity.

Each of the extracts of the above concentration was placed in a 96-well plate, and DPPH prepared in 100 uM methanol solution was added thereto so that the total volume of the solution was 200 μl.

After leaving this at 37° C. for 30 minutes, absorbance was measured at 560 nm.

The results are shown in Table 1 and FIG. 2 .

<Equation>

Free radical scavenging activity (%) = {100-(B/A)}X100

A: Absorbance of control wells not treated with the sample of the present invention

B: Absorbance of the wells of the experimental group treated with the sample of the present invention

Experimental Example 2: Cytotoxicity of the sample

In order to confirm the cytotoxicity of each mixture prepared in Examples 1 and 2, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed.

HDF (Human deraml fibroblast), B16F0, RAW 264.7 cells were aliquoted in a 96-well plate at 1 × 104 / well and stabilized for 18 hr in a 37%, 5% CO2 humidified incubator.

After that, the sample was treated with 5% and reacted for 20 hr.

All the medium was removed and 0.5 mg/ml MTT reagent was treated and reacted for 1 hr.

After removing the MTT solution, 200 ul of DMSO was added to dissolve formazan crystals, and absorbance was measured at 590 nm using a spectrophotometer.

The results are shown in Tables 2 and 3 for HDF (Human deraml fibroblast) cells, Tables 3 and 4 for B16F0 cells, and Tables 4 and 5 for RAW 264.7 cells.

Experimental Example 3: Measurement of inhibition of NO production

In order to confirm the anti-inflammatory activity of each extract obtained in Examples 1 and 2, the effect on NO production, which is known to play an important role in inducing inflammation, was investigated.

Nitric oxide was measured as nitrite and nitrate in the amount of NO in the cell supernatant.

For nitrite, a safe form of griess reagent after reduction with nitrate was used. When the confluence of RAW264.7 cells is 80% in a 96 well plate (5 × 10⁴cells/well), after washing twice with PBS, serum-free medium After incubation for more than 18 hours using

After taking the supernatant of the culture medium and reacting it with griess reagent, the absorbance was measured at 540 nm with an ELISA reader, and the NO production rate was expressed as a percentage.

In order to check whether the production of NO, a representative factor of the inflammatory response, is inhibited, RAW264.7 cells were treated with 1 μg/mL of lipopolysaccharide (LPS), and then the extracts were treated for each concentration 2 hours later.

After incubation for 18 hours, the amount of NO in the culture was measured.

The results are shown in Table 5 and FIG. 6 .

Experimental Example 4: Melanin assay measurement

In order to confirm the melanin production inhibitory effect of each mixture prepared in Examples 1 and 2, a melanin assay was performed.

B16F0 (melanocyte) cells were dispensed in a 96-well plate at a concentration of 2.5 × 10 ³ cells/well, stabilized in a 37%, 5% CO ₂ humidified incubator for 24 hr, and samples were treated for each concentration and reacted for 2 hr.

After the reaction, the stimulant, α-MSH, was diluted to a final concentration of 100 nM and treated.

The amount of melanin released into the medium after 72 hours was calculated by measuring the absorbance at 405 nm using a spectrophotometer.

The results are shown in Table 6 and FIG. 7 .

Experimental Example 5: Measurement of MMP-1

Real-time PCR was performed to confirm the mRNA of MMP-1 in each mixture prepared in Examples 1 to 2.

HDF (Human deraml fibroblast) cells were aliquoted into 3 × 10 ⁵ / 60 mm culture dishes, stabilized in a 37%, 5% CO ₂ humidified incubator for 18 hr, and then replaced with a serum-free medium.

5% of the sample was then treated. After 2 hours, UV-B was irradiated with 10 mJ/cm ² and reacted for 20 hr. After the reaction, the cells were harvested using NucleoZOL lysis buffer (NM, 740404), RNA isolation was performed using the protocol provided by MN, and the isolated RNA was quantified using a microplate reader, followed by HiSenScript™ RH ( -) cDNA was synthesized using the RT PreMix Kit (intron, 25087).

After performing real-time PCR using the synthesized cDNA and 2X Real-Time PCR Master Mix & Premix (BioFACT), the amplification products were quantitatively analyzed. The MMP-1 and β-actin primer sequences used for PCR are shown in Table 7.

The results are shown in Table 8 and FIG. 8 .

Example 3: Preparation of shampoo containing the extract of the present invention

Ammonium laureth sulfate 5g, cosylbetaine 5g, alkyl ether sulfate 0.5g, silicone 0.5g, polyquater 0.2g, fragrance 0.2g, and preservative 0.02g were mixed with 95% ethanol 5g.

5 g of the extract of the present invention obtained in Example and 5 g of glycerin were dissolved in 85.33 g of purified water, and the mixture was added thereto, followed by stirring to obtain a shampoo.

Example 4: Preparation of lotion containing extract of the present invention

To 8 g of 95% ethanol, 0.05 g of polypyrrolidone, 0.1 g of oleyl alcohol, 0.2 g of polyoxyethylene monooleate, 0.2 g of fragrance, 0.1 g of methyl paraoxybenzoate, a small amount of antioxidant, and a small amount of pigment were mixed.

10 g of the extract of the present invention obtained in Examples and 5 g of glycerin were dissolved in 85.33 g of purified water, and the mixture was added thereto, followed by stirring to obtain a lotion.

Example 5: Preparation of emulsion containing extract of the present invention

1.2 g of cetyl alcohol, 10 g of squalane, 2 g of vaseline, 0.2 g of paraoxybenzoic acid ethyl ester, 1 g of glycerin monoesterade, 1 g of polyoxyethylene (20 mol addition) monooleate and 0.1 g of incense furnace were heated and mixed at 70 ° C. After dissolving, 0.1 g of the extract of the present invention obtained in Examples, 5 g of dipropylene glycol, 2 g of polyethylene glycol-1500, 0.2 g of triethanolamine, and 76.6 g of purified water were heated to 75° C. and dissolved.

Both were mixed and emulsified, followed by cooling to obtain an oil-in-water (O/W) type emulsion.

Example 6: Preparation of a cosmetic solution containing the extract of the present invention

95% ethyl alcohol 5g, polyoxyethylene sorbitan monooleate 1.2g, kitulose 0.3g, sodium hyaluronate 0.2g, vitamin E-acetate 0.2g, sodium licorate 0.2g, paraoxybenzoate ethyl ester 0.1g, practice 1 g of the extract of the present invention obtained in Example and an appropriate amount of a pigment were mixed to obtain a cosmetic solution.

From the above description, those skilled in the art to which the present application pertains will be able to understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present application should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts thereof.

Korea Institute of Bioscience and Biotechnology KCTC14478BP 20210222

<110> HANKOOK COSMETICS MANUFACTURING CO., LTD. <120> Fermented extract for antioxidant, anti-inflammatory, whitening, and anti-wrinkle using Lactobacillus kunkeei Hankook-001strain, and manufacturing method of it, and cosmetic composition containing fermented extract <130> Hankook-001 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 871 <212> RNA <213> Unknown <220> <223> Lactobacillus kunkeei <400> 1 ctggcggcgt gcctaataca tgcaagtcga acgagctctc ccaaattgat tttatgcttg 60 cataaatgat ttttggattc ggagcgagtg gcgaactggt gagtaacacg tgggtaacct 120 gccccgaagc gggggataac atttggaaac aagtgctaat accgcataat tagttggaac 180 cgcatggttc caacttgaaa gatggctctg ctatcacttt gggatggacc cgcgccgtat 240 tagttagttg gtgagataaa agcccaccaa gacgatgata cgtagccgac ctgagagggt 300 aatcggccac attgggactg agacacggcc cagactccta cgggaggcag cagtagggaa 360 tcttccacaa tggacgaaag tctgatggag caacgccgcg tgagtgatga aggttttcgg 420 atcgtaaaac tctgttgtta aagaagaaca agtgttagag taactgttaa cactttgacg 480 gtatttaacc agaaagccac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg 540 caagcgttgt ccggatttat tgggcgtaaa gcgagcgcag gcggttttgt aagtctgctg 600 tgaaagccct cagctcaact gaggaagtgc agtggaaact acaaaacttg agtacagaag 660 aggaaagtgg aactccatgt gtagcggtga aatgcgtaga tatatggaag aacaccagtg 720 gcgaaggcgg ctttctggtc tgttactgac gctgaggctc gaaagcatgg gtagcgaaca 780 ggattagata ccctggtagt ccatgccgta aacgatgaat actaggtgtt ggagggtttc 840 cgcccttcag tgccgcagct aacgcattaa g 871 <210> 2 <211> 734 <212> RNA <213> Unknown <220> <223> Lactobacillus kunkeei <400> 2 aggtgttgga gggtttccgc ccttcagtgc cgcagctaac gcattaagta ttccgcctgg 60 gggatacgac cgcaaggttg aaactcaaag gaattgacgg gggcccgcac aagtggtgga 120 gcatgtggtt taattcgatg ctacgcgaag aaccttacca gctcttgaca tcttctgcca 180 acccaagaga ttgggcgttc ccttcgggga cagaatgaca ggtggtgcat ggttgtcgtc 240 agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt attattagtt 300 gccagcattt agttgggcac tctagtgaga ctgccggtga taaaccggag gaaggtgggg 360 acgacgtcaa atcatcatgc cccttatgag ctgggctaca cacgtgctac aatggatggt 420 acaacgagtc gcgaaaccgc gaggtcaagc taatctctta aagccattct cagttcggat 480 tgcaggctgc aactcgcctg catgaagttg gaatcactag taatcgtgga tcagcatgcc 540 acggtgaata cgttcccggg ccttgtacac accgcccgtc acaccatgag agtttgtaac 600 acccaaagac gatggggtaa ccttttagga gctagtcgtc taaggtggga cagatgatta 660 gggtgaagtc gtaacaaggt agccgtagga gaacctgcgg ctggatcacc ttcctttcta 720 cctggatctc ctcc 734

Claims (5)

In the method for producing a fermented extract for antioxidant, anti-inflammatory, whitening, and anti-wrinkle,
100 parts by weight of cultured wild ginseng root obtained by tissue culture and 1,000 parts by weight of purified water were put into an extraction container and mixed, followed by extraction at 90° C. for 3 hours to obtain an extract,
After the extract was sterilized at 121 ° C. for 15 minutes, allowed to cool, and Lactobacillus kunki Hankook-001 of accession number KCTC 14478BP was inoculated into the cooled extract and cultured and fermented at 35 ° C., 150 rpm, 2 days, and the fermentation broth was After removing the sediment and lactic acid bacteria by centrifugation at 10,000 rpm for 20 minutes, characterized in that the fermentation extract is prepared,
The culture environment conditions of the Lactobacillus kunki Hankook-001 strain of the accession number KCTC 14478BP are,
It is characterized in that the pH of the culture medium is 5.8, the incubation time is 44 hours, and the culture temperature is 32°C,
The fermented extract is
A method for producing a fermentation extract for antioxidant, anti-inflammatory, whitening, and anti-wrinkle using Lactobacillus kunki Hankook-001 strain, characterized in that it is contained in an amount of 0.01 to 10% by weight based on the total content of the cosmetic composition.
The method of claim 1,
The formulation of the cosmetic composition is,
Basic cosmetic formulations consisting of shampoo, lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O/W) type and water-in-oil (W/O) type;
a makeup base, foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, cheek color, and eyebrow pencil of a water-in-water or water-in-oil type makeup base; characterized in that any one selected from
Method for producing a fermentation extract for antioxidant, anti-inflammatory, whitening, and anti-wrinkle using Lactobacillus kunki Hankook-001 strain.
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